HO-endonuclease belongs to the dodecapeptide family of endonucleases that cleave a large, unique (&gt;18 bp) DNA recognition sequence that lacks dyad symmetry. These nucleases are encoded by nuclear or mitochondrial and chloroplast group I intron genes, or exist as inteins having their coding sequences embedded in-frame within unrelated genes that undergo protein splicing, such that two proteins are generated from a single translation product. Dodecapeptide endonucleases are known in phylogenetically diverse species, and new genes are constantly being discovered.
HO-endonuclease of the yeast Saccaromyces cerevisiae initiates a mating type switch by making a site-specific double strand break in the mating type gene MAT, and attains site-specificity by virtue of a large (18-24 bp) target site. It has now been shown that a 113-residue N-terminal truncation of HO-endonuclease is able to cleave its cognate site and to initiate a mating type switch in yeast.
HO is the only dodecapeptide endonuclease with a zinc finger DNA-binding domain. The present inventors have cloned an inactive allele of HO-endonuclease from a laboratory strain of yeast [Meiron, et al., "Identification of the Heterothallic Mutation in HO-endonuclease of S. cerevisiae Using HO/ho Chimeric Genes," Curr. Genet., Vol. 28, pp. 367-373 (1995)]. In that study, chimeric genes were made between the active and inactive endonuclease genes and tested for their activity in yeast. It was found that of the four amino acid substitutions in the mutant (ho) gene, only one substitution affected endonucleolytic activity. This mutation is in the first zinc finger of HO.
In the present invention, it is shown that it is possible to target endonucleolytic activity to a different site by exchange of the zinc finger domain of HO for that of the yeast transcription factor Swi5. It was found that a chimeric endonuclease comprising the nuclease domain of HO and the zinc finger domain of Swi5 cleaves the Swi5 target site URS1. This shows that it is possible to target the catalytic domain of dodecapeptide endonucleases via various DNA-binding moieties, and that they present a general nuclease domain in the design of highly site-specific targeted endonucleases.